Production of chitosan and chitin

ABSTRACT

The invention relates to a method of producing chitin or chitosan by culturing a  Rhizopus azygosporus  fungus or an  Actinomucor taiwanensis  fungus and isolating chitosan or chitin from the culture.

BACKGROUND OF THE INVENTION

Chitin is a highly insoluble N-acetylated polymer ofβ-(1,4)-D-glucosamine. Chitosan is an acid-soluble deacetylated form ofchitin. Chitin, chitosan, and derivatives thereof are used in a numberof industrial applications, including the production of viscositycontrol agents, adhesives, chromatography carriers, paper-strengtheningagents, flocculent agents, food additives, drugs, and cosmetics.

Chitin can be manufactured by the deproteination and decalcification ofcrab or shrimp shells. Chitosan can then be obtained by deacetylatingchitin with a hot alkali solution. This chitosan production process hasa number of unfavorable characteristics. For example, the processrequires expensive heat energy and caustic alkali, which is a potentialhealth hazard. The process also produces large amounts of waste, therebynecessitating significant disposal costs. In addition, the supply ofshrimp or crab shells is highly dependent upon seasonal andenvironmental factors, leading to unpredictable limitations onproduction capacity.

SUMMARY OF THE INVENTION

The invention is based on the discovery that unexpectedly high yields ofchitosan and chitin can be produced from the fungus Actinomucortaiwanensis and from the fungus Rhizopus azygosporus.

Accordingly, the invention features a method of producing chitosan orchitin by (1) culturing a Rhizopus azygosporus fungus or an Actinomucortaiwanensis fungus in a medium to form a culture, and (2) isolatingchitosan or chitin from the cells. For example, chitosan and chitin canbe isolated from the culture by separating the fungal cells from theculture and isolating the chitosan or chitin from the separated cells.

The invention also includes a method of producing chitosan or chitin by(1) culturing a fungus of the family Mucoraceae in a medium useful inthe methods of the invention to form a culture, and (2) isolatingchitosan or chitin from the fungal culture.

A medium useful for the methods of the invention can include about 5 to60 g/L (e.g., about 30 g/L) corn steep liquor, about 10-100 g/L (e.g.,about 50 g/L) glucose, about 0.01 to 30 g/L (e.g., about 2.5 g/L)ammonium sulfate, or other suitable ingredients.

The methods of the invention allow surprisingly high-yield production ofchitosan or chitin from a culture containing a Rhizopus azygosporus orActinomucor taiwanensis fungus. Further, it has been discovered that amedium containing corn steep liquor, glucose, yeast extract, andammonium sulfate is capable of increasing the output of chitin andchitosan from a fungal culture. The methods of the invention, therefore,provide an alternative to producing chitin and chitosan without relianceon environmentally harmful chemicals or the variable abundance of thecrustacean crop.

Other features or advantages of the present invention will be apparentfrom the following detailed description, and also from the claims.

DETAILED DESCRIPTION

The invention relates to high yield production of chitosan or chitinfrom fungal cultures belonging to the family Mucoraceae.

Particular fungi useful in the methods of the invention include Rhizopusazygosporus and Actinomucor taiwanensis. Both of these organisms areavailable upon request from the Culture Collection and Research Center(CCRC), Food Industry and Research Development Institute, No. 331,Shih-Ping Road, Hsinchu 300, Taiwan, Republic of China. R. azygosporusis available as Catalog No. CCRC31558, and A. taiwanensis is availableas Catalog No. CCRC31559.

Procedures for culturing fungi are well known in the art. For example,YM agar can be inoculated with a fungus, and the inoculated agarincubated at 25° C. to 37° C. for 3 to 6 days. Spores obtained from thefungus are suspended in liquid to achieve a 10⁴ to 10⁷ cfu/ml stock.This stock is directly inoculated into a fermentation medium.

The fermentation medium can have an initial pH ranging from 3 to 8 andcan contain 10 to 100 g/L of a carbon source (e.g., glucose, sucrose,corn starch, molasses, or soybean oil), 5 to 60 g/L of a nitrogen source(e.g., soybean meal, peptone, or corn steep liquor), 0.5 to 20 g/L ofyeast extract, 0.01 to 30 g/L (NH₄)₂SO₄, 0 to 3 g/L K₂HPO₄, 0 to 3 g/LNaCl, 0 to 15 g/L MgSO₄.7H₂O, and/or 0 to 0.3 g/L CaCl₂. The fungus isgrown in the fermentation medium for an additional two to four days.

Chitosan can be isolated and purified from fungal mycelia by standardmethods. For example, alkaline and acid treatment can be used to isolatechitosan as described in McGahren et al., Process Biochem 19:88-90,1984. Additional details and procedures for isolating chitosan can befound in European Application No. 0531991 A2; Yokoi et al., J FermenBioeng 85:246-249, 1998; U.S. Pat. No. 5,232,842,; Rane et al., FoodBiotech 7:11-33, 1993; and Hang, Biotech Lett 12:911-912, 1990.

In general, the cell mass is separated from the fermentation broth andwashed with distilled water. The cells are then treated with 0.5 to 2 NNaOH, and the alkaline mixture incubated at 121° C. for 15 minutes. Thesolid material is then pelleted by centrifugation and washed withdistilled water and ethanol. The washed material is treated with a 2%acetic acid solution and incubated at 95° C. for 12 hours. The resultingslurry is then isolated by centrifugation, yielding an acid-solublesupernatant (containing chitosan) and an acid-insoluble precipitate(containing chitin).

The pH of the supernatant is adjusted to 10 with 2 N NaOH, therebyprecipitating out the chitosan. The chitosan is finally washed withdistilled water and freeze-dried. The acid-insoluble precipitate is alsowashed with distilled water and freeze-dried. This acid-insoluble andalkali-insoluble fraction is purified chitin.

Without further elaboration, it is believed that one skilled in the artcan, based on the above disclosure and the description below, utilizethe present invention to its fullest extent. The following examples areto be construed as merely illustrative of how one skilled in the art canpractice the invention and are not limitative of the remainder of thedisclosure in any way. Any publications cited in this disclosure arehereby incorporated by reference.

The results for the following examples are summarized in Table 2, whichappears after the Examples.

EXAMPLE 1

Spore suspension of Rhizopus azygosporus from 4-day slant cultures wasinoculated directly into 250 ml shaker flasks containing 100 ml of freshfermentation medium. Fermentation was carried out at 28° C. for 48hours, while shaking at 200 rpm. Each liter of medium contained 10 g ofa nitrogen source (soybean meal, peptone, or corn steep liquor), 20 g ofa carbon source (glucose or corn starch), 1 g yeast extract, 5 g(NH₄)₂SO₄, 1 g K₂HPO₄, 1 g NaCl, 5 g MgSO₄.7H₂O, and 0.1 g CaCl₂.

The cell mass was recovered from the fermented broth and treated with 1N NaOH at 121° C. for 15 minutes. The alkali-insoluble material wassuspended in 2% acetic acid, and the mixture incubated at 95° C. for 12hours to solubilize the chitosan. Chitosan was precipitated by adjustingpH of the acid-soluble supernatant to 10. The chitosan was then washed,dried, and weighed. The acid-insoluble material, chitin, also waswashed, dried, and weighed. Yields were calculated in terms of gramschitin or chitosan per liter of culture, and the results are presentedin Table 2. The highest yields of chitin and chitosan were achievedusing the medium containing corn starch and peptone.

EXAMPLE 2

A spore suspension of Actinomucor taiwanensis from 4-day slant cultureswas inoculated directly into 250 ml shaker flasks containing 100 ml offermentation medium. Fermentation and chitosan and chitin isolation wasperformed as described in Example 1, and the results presented in Table2. High yields of chitin and chitosan could be achieved using the mediumcontaining corn steep liquor and glucose.

EXAMPLE 3

R. azygosporus was cultured and processed as described in Example 1above, except that all media contained peptone as the nitrogen source,and the source of carbon was varied among glucose, corn starch, sucrose,molasses, and soybean oil. Inclusion of corn starch led to a high yieldof 0.9 g/L for both chitosan and chitin, while inclusion of soybean oilled to the highest yield of 1.5 g/L for chitin for this Example (Table2).

EXAMPLE 4

A. taiwanensis was cultured and processed as described in Example 2above, except that all media contained corn steep liquor as the nitrogensource, and the source of carbon was varied among glucose, corn starch,sucrose, molasses, and soybean oil. Inclusion of glucose as the carbonsource led to the best yield of both chitin and chitosan for thisExample (Table 2).

EXAMPLE 5

R. azygosporus was cultured and processed as described in Example 1,except that each liter of medium contained 30 g corn steep liquor, 50 gglucose, 2 g yeast extract, 2.5 g (NH₄)₂SO₄, and 0.05 g CaCl₂. Thisculture led to a high yield of 1.1 g/L for chitosan (Table 2).

EXAMPLE 6

A. taiwanensis was cultured and processed as described in Example 2,except that each liter of medium contained 30 g corn steep liquor, 50 gglucose, 2 g yeast extract, 2.5 g (NH₄)₂SO₄, and 0.05 g CaCl₂. Thisculture led to the highest combined yield of chitosan (1.7 g/L) andchitin (1.1 g/L) (Table 2).

EXAMPLE 7

A. taiwanensis was cultured and processed as described in Example 6,except that the medium contained 0.5 g K₂HPO₄ per liter instead ofCaCl₂. This culture led to a high yield of 1.4 g/L for chitosan.

TABLE 2 EXAM- PLE/ Nitrogen Source Carbon Source Salt Yield (g/L) strainS.M. peptone C.S.L. glucose C.S. sucrose molasses S.O. (NH4)₂SO₄ K₂HPO₄NaCl MgSO₄ CaCl₂ chitosan chitin 1/R.a. X X X X X X X 0.3 0.9 X X X X XX X 0.7 0.6 X X X X X X X 0.5 0.7 X X X X X X X 0.2 0.7 X X X X X X X0.9 0.9 X X X X X X X 0.4 0.8 2/A.t. X X X X X X X 0.5 0.4 X X X X X X X0.7 0.6 X X X X X X X 0.9 0.9 X X X X X X X 0.5 0.8 X X X X X X X 0.61.0 X X X X X X X 0.6 0.6 3/R.a. X X X X X X X 0.7 0.6 X X X X X X X 0.90.9 X X X X X X X 0.4 0.7 X X X X X X X 0.8 0.6 X X X X X X X 0.5 1.54/A.t. X X X X X X X 0.9 0.9 X X X X X X X 0.6 0.6 X X X X X X X 0.6 0.4X X X X X X X 0.3 0.3 X X X X X X X 0.2 0.7 5/R.a. X X X X 1.1 0.66/A.t. X X X X 1.7 1.1 7/A.t. X X X X 1.4 0.7

The abbreviations used in Table 2 are as follows: “R.a.” stands forRhizopus azygosporus; “A.t.” stands for Actinomucor taiwanensis; “S.M.”stands for soybean meal; “C.S.L.” stands for corn steep liquor; “C.S.”stands for corn starch; and “S.O.” stands for soybean oil. All culturesdescribed in Table 2 contained yeast extract.

The results in Examples 1-7 above indicated that (1) A. taiwanensis andR. azygosporus are superior producers of chitin and chitosan, and (2)media containing corn steep liquor, glucose, and ammonium sulfate canincrease the yield of chitin and chitosan produced by fungi.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thisinvention.

What is claimed is:
 1. A method of producing chitosan or chitin, themethod comprising culturing an Actinomucor taiwanensis fungus in amedium to form a culture, and isolating chitosan or chitin from theculture, wherein the medium comprises about 5 to 60 g/L corn steepliquor.
 2. The method of claim 1, wherein the medium comprises about 30g/L corn steep liquor.
 3. The method of claim 1, wherein the mediumfurther comprises 0.01 to 30 g/L ammonium sulfate.
 4. The method ofclaim 3, wherein the medium comprises about 2.5 g/L ammonium sulfate. 5.The method of claim 1, wherein the medium further comprises about 10-100g/L glucose.
 6. The method of claim 5, wherein the medium comprisesabout 50 g/L glucose.
 7. The method of claim 1, further comprisingseparating fungal cells from the culture, and wherein the chitosan orchitin is isolated from the separated fungal cells.
 8. A method ofproducing chitosan or chitin, the method comprising culturing anActinomucor taiwanensis fungus in a medium to form a culture, andisolating chitosan or chitin from the culture, wherein the mediumcomprises about 10-100 g/L glucose.
 9. The method of claim 8, whereinthe medium comprises about 50 g/L glucose.
 10. The method of claim 8,wherein the medium further comprises about 0.01 to 30 g/L ainmoniunsulfate.
 11. The method of claim 10, wherein the medium furthercomprises about 2.5 g/L ammonium sulfate.
 12. The method of claim 8,further comprising separating fungal cells from the culture, and whereinthe chitosan or chitin is isolated from the separated fungal cells.